Wang Yong-Xiang, Zhang An-Yun, Yang Yong-Qiang, Lei Chang-Wei, Cheng Guang-Yang, Zou Wen-Cheng, Zeng Jin-Xin, Chen Yan-Peng, Wang Hong-Ning
Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610065, Sichuan, PR China; Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, Chengdu 610065, Sichuan, PR China.
Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610065, Sichuan, PR China; Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, Chengdu 610065, Sichuan, PR China; "985 Project" Science Innovative Platform for Resource and Environment Protection of Southwestern, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, Sichuan University, Chengdu 610065, Sichuan, PR China.
J Microbiol Methods. 2018 Oct;153:24-30. doi: 10.1016/j.mimet.2018.08.003. Epub 2018 Aug 9.
Salmonella enterica serovar Indiana (S. Indiana) was the most frequently reported foodborne pathogen, which has a broad host range including poultry, swine, and humans. Traditional methods used for the detection of S. Indiana from contaminated food products are time-consuming and labor-intensive. Therefore, rapid detection methods with high sensitivity and specificity are vitally important to prevent the spread of S. Indiana. In this study, we developed a nearly instrument-free, simple molecular method which incorporates cross-priming amplification (CPA) combined with a nucleic acid detection strip (NADS) for sensitive detection of S. Indiana. A set of CPA primers was designed based on S. Indiana specific nucleotide sequences and the specificity of CPA-NADS was tested against 42 bacterial strains. The results showed that this method was highly specific for detection of S. Indiana. The sensitivity of CPA-NADS was evaluated and compared with that of the serovar-specific PCR method and the real-time PCR method. The limit of detection of the CPA method was 8.997 fg/μL for genomic DNA and 6.2 × 10 CFU/mL for bacteria in pure culture. An application of the CPA assay was conducted with 90 inoculated specimens by S. Indiana. The accuracy of CPA-NADS was consistent with the results of the traditional culture-based methods in inoculated specimens. This method showed a higher sensitivity than the serovar-specific PCR method did and was more convenient to perform. In conclusion, we demonstrated that the CPA-NADS system offers high specificity, sensitivity, rapidity, and a simple detection tool for screening S. Indiana.
肠炎沙门氏菌印第安纳血清型（S. Indiana）是报告频率最高的食源性病原体，其宿主范围广泛，包括家禽、猪和人类。从受污染食品中检测S. Indiana的传统方法既耗时又费力。因此，具有高灵敏度和特异性的快速检测方法对于预防S. Indiana的传播至关重要。在本研究中，我们开发了一种几乎无需仪器的简单分子方法，该方法将交叉引物扩增（CPA）与核酸检测条（NADS）相结合，用于灵敏检测S. Indiana。基于S. Indiana的特异性核苷酸序列设计了一组CPA引物，并针对42株细菌测试了CPA-NADS的特异性。结果表明，该方法对S. Indiana的检测具有高度特异性。评估了CPA-NADS的灵敏度，并与血清型特异性PCR方法和实时PCR方法进行了比较。CPA方法对基因组DNA的检测限为8.997 fg/μL，对纯培养细菌的检测限为6.2×10 CFU/mL。用90份接种了S. Indiana的标本进行了CPA检测的应用。CPA-NADS在接种标本中的准确性与传统培养方法的结果一致。该方法显示出比血清型特异性PCR方法更高的灵敏度，并且操作更方便。总之，我们证明了CPA-NADS系统为筛选S. Indiana提供了高特异性、灵敏度、快速性和简单的检测工具。
